Methods for providing neuroprotection for the animal central nervous system against neurodegeneration caused by ischemia

ABSTRACT

Methods and pharmaceutical compositions for preconditioning and/or providing neuroprotection to the animal central nervous system against the effects of ischemia, trauma, metal poisoning and neurodegeneration, including the associated cognitive, behavioral and physical impairments. In one embodiment, the method is accomplished by stimulating and stabilizing hypoxia-inducible factor-1α (HIF-1α). HIF-1α is known to provide a neuroprotective benefit under ischemic conditions. Patients at risk for certain diseases or disorders that are associated with risk for cerebral ischemia may benefit, e.g., those at risk for Alzheimer&#39;s disease, Parkinson&#39;s disease, Wilson&#39;s disease or stroke or those patients having head or spinal cord injury. Patients undergoing certain medical procedures that may result in ischemia may also benefit. Initially, the possibility of ischemia or neurodegeneration is recognized. Intranasal therapeutic agents are administered to the upper third of the nasal cavity to bypass the blood-brain barrier and access the central nervous system directly to avoid unwanted and potentially lethal side effects. Therapeutic agents include those substances that interact with iron and/or copper such as iron chelators, copper chelators, and antioxidants. A particular example of such therapeutic agents is the iron chelator deferoxamine (DFO). Intranasal administration of DFO is known to stimulate and/or stabilize HIF-1α and provides an efficient and safe method for pre-conditioning the brain to protect against cerebral ischemia. Moreover, DFO is shown to decrease weight loss in subjects when administered pre and/or post stroke.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application claiming the benefit of andpriority to U.S. provisional patent application No. 60/601,547 filedAug. 13, 2004, which is incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to methods for preconditioning and/orproviding neuroprotection to the animal central nervous system againstischemia, neurodegeneration, trauma and metal poisoning, includingassociated cognitive, behavioral and physical impairments.

2. Description of the Related Art

Certain medical procedures, for example coronary artery bypass graft(CABG) surgery, are associated with neurological complications. In thecase of CABG, the surgery is performed on more than 800,000 patientsworldwide each year. Many of the CABG procedures performed areassociated with neurological complications. These complications rangefrom stroke in up to 16% of the patients to general cognitive declinewith 50% of patients having impairment post-surgery and with progressivedecline occurring in some patients over the next five years. Inaddition, physical and behavioral impairment manifest in some CABGpatients. Newman M F et al., N. Eng. J. Med. 344:395-402 (2001);Brillman J., Neurol. Clin. 11:475-495 (1993); and Selnes, O. A., Ann.Thorac. Surg. 67:1669-1676 (1999) are instructive.

Originally, it was hypothesized that the neurological complicationsassociated with CABG surgery were either procedure or patient-related.The procedure generally implicated as potentially harmful wascardiopulmonary bypass using a pump and oxygenator. However, a recentstudy reports no difference in cognitive outcome between groups ofpatients undergoing CABG surgery performed with, or without, the pumpand oxygenator. Such results suggest that the neurological impairmentsfollowing CABG surgery may, in fact, be patient-related and, as aresult, amenable to therapeutic manipulation.

In addition, patients at risk for, or diagnosed with disorders involvingneurological impairments, e.g., Alzheimer's disease, Parkinson'sdisease, stroke, traumatic brain injury, spinal cord injury may benefitfrom similar therapeutic manipulation. See Crapper McLachlan, D. R.,Dalton, A. J., Kruck, T. P. A., Bell, M. Y., Smith, W. L., Kalow, W.,and Andrews, D. F. Intramuscular desferrioxamine in patients withAlzheimer's disease. The Lancet 337:1304-1308, 1991.

A number of neurodegenerative disorders are known to havemetal-associated pathology, i.e., resulting at least in part from metalpoisoning, and may benefit from the therapeutic manipulationcontemplated by embodiments of the present invention. These include AD,PD, Creutzfeldt-Jakob disease, familial amyotrophic lateral sclerosis,lewy-body dementia, carotid atherosclerosis, tardive dyskinesia,multiple sclerosis, Wilson's disease, progressive supranuclear palsy,Hallervorden-Spatz syndrome, multisystem atrophy, Huntington's disease,familial basal ganglia degeneration, Down's syndrome, cataracts,haemochromatosis, cerebral haemorrhage and head injury. See P. M.Doraiswamy and A. E. Finefrock, Metals in our minds: therapeuticimplications for neurodegenerative disorders, The Lancet Neurology, Vol.3, July 2004.

In general, ischemic conditions activate a number of genes that areimportant in the cellular and tissue adaptation to low oxygenconditions. These genes include erythropoietin, glucose transporters,glycolytic enzymes, and the vascular endothelial growth factor (VEGF).VEGF is a major angiogenic factor that has been shown to activate newblood vessel formation. Transcriptional up-regulation has been shown tobe implicated in the induction of the VEGF gene, an action mediated bythe specific binding of the hypoxia-inducible factor-1 (HIF-1) to thehypoxic response element (HRE).

The HIF-1 transcription factor is a heterodimer composed of HIF-1α andHIF-1β and regulates the adaptive response to hypoxia in animal cells.HIF-1α accumulates under hypoxic conditions, but is virtuallyundetectable in normal oxygen conditions. HIF-1β, on the other hand, isreadily found in all cells. The HIF-1 heterodimer is believed to beneuroprotective against ischemia through the activation of EPO and VEGF.

HIF-1α has been shown in vitro to be activated by metal chelators,including both iron and copper chelating agents. A particular example ofsuch an agent is deferoxamine (DFO), a hexadentate iron chelator, withkinetics similar to those associated with hypoxia, resulting inincreased expression of HIF-1 target genes, including EPO and VEGF. DFOis also known to stabilize HIF-1 subunits, possibly by chelating andinactivating the iron that plays a role in targeting the subunit forproeolytic degradation under normoxic conditions.

In vivo studies have demonstrated that DFO induces HIF-1α in neonataland adult rats, injecting the chelator either subcutaneously (s.c.) orintraperitoneally (i.p.), typically in very high dosage. In addition,studies indicate that the following substances stimulate and/orstabilize HIF-1α: insulin, IGF-I, heregulin insulin, heregulin, TGFbeta,IL-1beta, TNFalpha, cobalt, pyruvate, oxalacetate and lactate.

Problems exist, however, with the administration of DFO intravenously.DFO is not generally injected intravenously for at least two reasons.First, it is a small molecule and, as a result, is eliminated rapidlythrough the kidney. The typical plasma half-life in humans is less than10 minutes. Second, the injection of an intravenous bolus of DFO causesacute hypotension that is rapid, may lead to shock and may be lethal.These characteristics have limited the utility of DFO in particular as aneuroprotective agent.

One published study administered DFO intranasally to iron overloadedpatients. G. S. Gordon et al., Intranasal Administration of Deferoxamineto Iron Overloaded Patients, (1989) Am. J. Med. Sci. 297(5):280-284. Inthis particular study, DFO was administered to the patients as a nasalspray in a volume of 75 microliters per spray. Significantly, suchsprays are known to deposit the drug or other substance in the lowerthird of the nasal cavity. This is verified by patient observationsstating that a bad taste in the mouth was resulting from the drugpassing through the nasopharynx and into the mouth. As a result, thisstudy did not involve delivering the drug to the upper third of thenasal cavity. Thus, the drug would not have reached the olfactoryepithelium or the olfactory nerves. As a result, delivery of the drug tothe CNS would be less than optimal.

It is recognized that intranasal delivery to the CNS may occur alongboth the olfactory and trigeminal nerve pathways. See Thorne, R G(2004), Delivery of Insulin-Like Growth Factor-I to the Rat Brain andSpinal Cord Along Olfactory and Trigeminal Pathways Following IntranasalAdministration, Neuroscience, Vol. 127, pp. 481-496. Optimal deliverytaking advantage of both pathways is accomplished by administering thesubstance in the upper third of the nasal cavity.

Regarding Alzheimer's disease, some studies indicate that cerebralvascular problems occur first, followed by neurodegeneration in laterstages of the disease. For example, see The Lancet Neurology, vol. 3,page 184-190, Jack C. de la Torre (March, 2004). Thus, it may bepossible to prevent, mitigate or treat the effects of Alzheimer'sdisease at the appropriate disease stage through therapeuticmanipulation targeted toward mitigation or prevention of cerebralischemia or neurodegeneration.

In a published patent application, U.S. Pat. App. No. 20020028786 byWilliam H. Frey II (also a co-inventor of the present application)entitled METHODS AND COMPOSITIONS FOR ENHANCING CELLULAR FUNCTIONTHROUGH PROTECTION OF TISSUE COMPONENTS, various substances arediscussed that may be administered intranasally to treat variousdiseases and conditions. The entire contents of this reference arehereby incorporated by reference.

BRIEF SUMMARY OF THE INVENTION

Given the situation described above there is a need for a method forefficiently and safely conditioning, or preconditioning, the animal CNSto prevent or minimize cognitive, behavioral and physical impairment dueto ischemia, neurodegeneration, CNS trauma and free radical damage fromcopper, zinc and iron. In addition, there is a need for a method forefficiently and safely conditioning and treating the animal CNS to treatexisting cognitive, behavioral and physical impairment due to ischemia,neurodegeneration, CNS trauma and free radical damage from copper andiron. Further, there is a need to optimize the administration ordelivery of a therapeutic agent.

Methods and pharmaceutical compositions for preconditioning the CNS toneuroprotect against, minimize and/or prevent the effects of ischemiaand the cognitive, behavioral and physical impairments that oftenaccompany ischemic episodes by stimulating and stabilizinghypoxia-inducible factor-1α (HIF-1α) are provided herein. HIF-1α isknown to provide a neuroprotective benefit under ischemic conditions.Patients at risk for certain diseases or disorders that carry aconcomitant risk for ischemia may benefit, e.g., those at risk forAlzheimer's disease, Parkinson's disease or stroke. Patients undergoingcertain medical procedures that may result in ischemia may also benefit.

In addition, methods and compounds for treating patients that haveundergone an ischemic episode to minimize the effects of the ischemia.

Initially, under a representative embodiment of the invention, thepossibility of an ischemic episode or neurodegeneration is recognized.Intranasal therapeutic agent is administered to the upper third of thenasal cavity to bypass the blood-brain barrier and access the centralnervous system directly to avoid unwanted and potentially damaging sideeffects. Therapeutic agents include those substances that may interactwith iron and/or copper such as iron chelators, copper chelators, and/orantioxidants and free radical scavengers. A particular example of suchtherapeutic agents is the iron chelator deferoxamine (DFO). DFO may alsochelate copper as well as other metals. Intranasal administration of DFOis known to stimulate and/or stabilize HIF-1α and provides an efficientand safe method for pre-conditioning the CNS to protect against cerebralischemia. The effects of pretreating a patient, who ultimatelyexperiences ischemia, include significant reduction of infarct volume aswell as a significant decrease in weight loss following stroke.

Another embodiment of the invention provides a method and pharmaceuticalcompositions for treating a patient that has undergone an ischemicepisode by administering at least one does of a therapeutic agent viaintranasal delivery to the upper one-third of the nasal cavity. Asdiscussed above in connection with pretreatment, metal chelators, e.g.,DFO, may be used to treat a patient post-stroke. The effects ofpost-stroke intranasal administration of such a therapeutic agentinclude significant reduction of infarct volume as well as a significantdecrease in weight loss following stroke.

Thus, one embodiment of the invention relates to a method andpharmaceutical compositions for preventing, minimizing and/or treatingneurologic complications due to cerebral ischemia as a result of certainmedical procedures. The method comprises administering at least one doseof a therapeutic agent via intranasal delivery to the upper one-third ofthe nasal cavity prior to undergoing a medical procedure that may resultin neurologic complications.

Another embodiment of the invention relates to a method andpharmaceutical compositions for preventing, minimizing and/or treatingneurologic complications due to cerebral ischemia or neural degenerationas a result of certain medical procedures. The method comprisesadministering at least one dose of at least one therapeutic agent viaintranasal delivery to the upper one-third of the nasal cavity during,prior to and/or after undergoing a medical procedure that may result inneurologic complications. The particular therapeutic agent(s) selectedmay preferentially chelate iron or copper or a combination of the ironand copper, or otherwise interact with select metals or preventoxidation/reduction cycling of iron or copper.

Yet another embodiment of the invention relates to a method andpharmaceutical compositions for decreasing weight loss encountered bypatients having cerebral ischemic episode.

Another embodiment of the invention relates to a method andpharmaceutical compositions for preventing, minimizing and/or treatingneurologic complications due to cerebral ischemia and/orneurodegeneration for patients at risk for, or diagnosed with, certainmedical conditions such as Alzheimer's disease, Parkinson's disease,Creutzfeldt-Jakob disease, familial amyotrophic lateral sclerosis,lewy-body dementia, carotid atherosclerosis, tardive dyskinesia,multiple sclerosis, Wilson's disease, progressive supranuclear palsy,Hallervorden-Spatz syndrome, multisystem atrophy, Huntington's disease,familial basal ganglia degeneration, Down's syndrome, cataracts,haemochromatosis, cerebral hemorrhage, subarachnoic hemorrhage, headinjury, and spinal cord injury.

The figures and the detailed description which follow more particularlyexemplify these and other embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, which are as follows.

FIG. 1 is a bar graph illustrating pre-stroke treatment with intranasaladministration of three doses of 10% DFO and its effect on infarctvolume.

FIG. 2 is a bar graph illustrating pre-stroke treatment with intranasaladministration of one dose of 10% DFO and its effect on infarct volume.

FIG. 3 is a bar graph illustrating pre-stroke treatment with intranasaladministration of three doses of 3% DFO and its effect on infarctvolume.

FIG. 4 is a bar graph illustrating post-stroke treatment with intranasaladministration of six doses of 10% DFO and its effect on infarct volume.

FIG. 5 is a bar graph illustrating pre-stroke treatment with intranasaladministration of one dose of 10% DFO and its effect on weight losspost-stroke.

FIG. 6 is a bar graph illustrating pre-stroke treatment with intranasaladministration of three doses of 3% DFO and its effect on weight losspost-stroke.

FIG. 7 is a bar graph illustrating pre-stroke treatment with intranasaladministration of three doses of 10% DFO and its effect on weight losspost-stroke.

FIG. 8 is a bar graph illustrating post-stroke treatment with intranasaladministration of six doses of 10% DFO and its effect on weight losspost-stroke.

DETAILED DESCRIPTION OF THE INVENTION, INCLUDING THE BEST MODE

While the invention is amenable to various modifications and alternativeforms, specifics thereof are shown by way of example in the drawings anddescribed in detail herein. It should be understood, however, that theintention is not to limit the invention to the particular embodimentsdescribed. On the contrary, the intention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention.

Definitions

As used herein, “central nervous system” (CNS) refers to the brain andspinal cord and associated tissues.

An “effective amount” of agent is an amount sufficient to prevent,treat, reduce and/or ameliorate the symptoms, neuronal damage and/orunderlying causes of any of the referenced disorders or diseases. Insome instances, an “effective amount” is sufficient to eliminate thesymptoms of those diseases and overcome the disease itself.

In the context of the present invention, the terms “treat” and “therapy”and “therapeutic” and the like refer to alleviate, slow the progression,prophylaxis, attenuation or cure of ischemia, trauma, metal poisoning orneurodegeneration.

“Prevent”, as used herein, refers to putting off, delaying, slowing,inhibiting, or otherwise stopping, reducing or ameliorating the onset ofischemia, trauma, metal poisoning or neurodegeneration. It is preferredthat a large enough quantity of the agent be applied in non-toxic levelsin order to provide an effective level of neuroprotection. The method ofthe present invention may be used with any animal, such as a mammal or abird (avian), more preferably a mammal. Poultry are a preferred bird.Exemplary mammals include, but are not limited to rats, mice, cats,dogs, horses, cows, sheep, pigs, and more preferably humans.

Thus, methods and pharmaceutical compositions are described herein that,inter alia, prevent, and/or treat neurologic complications such ascognitive, behavioral and/or physical impairment due to ischemia,neurodegeneration, trauma and metal poisoning.

An alternative to potentially lethal and generally ineffectiveintravenous injection of metal chelators, e.g., DFO, may be accomplishedusing an alternative non-invasive method to directly target thesubstance to the brain and thus the central nervous system (CNS).Intranasal delivery allows substances to be rapidly delivered to thecentral nervous system, even those that do not readily cross theblood-brain barrier by bypassing the blood-brain barrier and directlyexposes the CNS to the delivered substance. In this manner, unwantedsystemic side effects are reduced if not eliminated.

Since DFO, similar to other metal chelators, has a strong Fe-III bindingconstant (10³¹), it is rapidly eliminated from the blood and does notreadily cross the blood-brain barrier. Thus, when metal chelator-basedtherapeutic agents are administered intravenously, orally or evenintranasally—but not directly to the upper one-third of the nasalcavity—to target affected tissues within the brain, the therapeuticeffect has been heretofore minimal. Delivery of intranasal DFO to theupper one-third of the nasal cavity has been assessed by administering 6mg DFO bound to 6 μCi of ⁵⁹Fe (as ⁵⁹FeCl₃) to rats under anesthesia. TheIN dose in 60 μL was administered as 6 μL drops over twenty minutes.Following delivery, tissues were removed for analysis. Usingscintillation counting, labeled ferrioxamine was detected throughout thebrain, with high concentrations detected in the olfactory bulbs,anterior olfactory nucleus, hypothalamus, frontal cortex and cervicalspinal cord. Even higher ferrioxamine concentrations were observed inthe trigeminal nerves and ventral dura. Peripheral tissues with thehighest ferrioxamine concentrations included the olfactory epithelium,thyroid and cervical lymph nodes. By contrast, the blood concentrationsof ferrioxamine, taken at 5 minute intervals from dosing up to 25minutes post-dose, are quite low, indicating a minimization of exposureof the therapeutic agent to non-target tissue. The data provided inTable 1 below, thus illustrates that intranasal DFO, the concentrationshaving been calculated based on an extrapolation of the ferrioxamineconcentration, administered to the upper one-third of the nasal cavity,is effectively delivered to the brain and upper spinal cord, withminimal systemic exposure.

TABLE 1 Intranasal Delivery of DFO (uM concentrations in tissues @ 25minutes after the onset of delivery) uL delivered 62 65 60 60 64 62 6262 66 61 uCi delivered 36.55 38.40 35.45 35.35 36.77 35.28 35.30 34.7235.80 34.31 mg delivered 6.15 6.44 5.95 5.95 6.29 6.05 6.05 6.07 6.456.00 nmol delivered 9,361.73 9,801.65 9,063.49 9,053.64 9,583.979,218.26 9,207.99 9,237.98 9,824.75 9,128.91 Drug Delivery Time 21 21 2018 20 22 20 20 20 18 Time of Perfusion 25 25 26 27 25 26 27 26 26 26 Ratweight 303 302 264 281 298 309 336 283 318 315 RAT # DF09 DF10 DF11 DF12DF13 DF14 DF15 DF18 DF19 DF20 Blood Sample 1 (5:00) 1.2 1.6 0.6 1.2 0.71.5 1.1 0.8 0.3 1.8 Blood Sample 2 (10:00) 1.1 2.1 1.1 1.2 1.2 1.8 1.71.0 0.4 1.9 Blood Sample 3 (15:00) 1.1 2.0 0.5 1.8 0.9 1.4 1.7 1.3 0.52.6 Blood Sample 4 (20:00) 1.1 1.8 0.3 1.9 1.1 1.6 1.5 1.1 0.4 2.9 BloodSample 5 (25:00) 1.8 1.6 1.8 1.3 1.5 2.2 1.7 1.3 0.5 2.1 SuperficialNodes (4) 3.4 0.9 0.6 0.9 2.2 0.6 1.8 0.6 1.1 0.8 Cervical Nodes (2)12.9 10.9 34.2 40.8 58.2 51.4 65.1 13.2 11.4 8.1 Dorsal Dura 26.5 11.47.4 14.1 16.6 32.0 8.0 5.9 35.8 5.1 Ventral Dura 25.3 38.7 70.9 17.758.3 44.0 51.5 — 62.8 11.6 Trigeminal Nerve 33.3 14.7 22.4 8.4 72.8 25.126.6 17.4 27.0 9.5 Olfactory Bulbs 12.7 10.6 30.0 14.7 20.5 13.1 28.027.5 21.6 6.6 Anterior Olfactory Nucleus 4.4 4.2 — — 5.4 2.5 5.5 4.4 7.7— Frontal Cortex 4.3 3.3 13.6 — 2.5 1.1 6.5 1.4 5.0 — Caudate/Putamen2.0 1.5 2.1 — 2.4 0.9 1.6 1.1 2.0 — Septal Nucleus 2.6 1.6 1.6 — 3.2 1.92.0 1.8 2.9 — Hippocampus 0.9 0.9 0.9 — 2.3 1.2 1.2 0.5 1.3 — Parietalcortex 1.3 1.6 2.3 — 0.7 1.9 2.8 0.8 1.0 — Thalamus 1.1 1.2 1.2 — 1.51.0 1.0 0.8 1.2 — Hypothalamus 5.4 7.3 6.5 — 3.1 3.0 6.1 2.7 3.8 —Midbrain 1.3 1.3 1.1 — 1.8 1.3 1.2 0.6 1.3 — Pons 2.0 1.5 1.4 — 1.5 2.02.6 0.7 2.4 — Medulla 1.1 2.3 1.2 — 1.7 2.2 3.0 1.0 2.0 — Upper CervicalSpinal Cord 2.1 1.4 3.7 1.5 3.9 6.8 7.3 1.4 4.6 4.6 Cerebellum 0.8 0.90.6 — 0.9 1.4 1.1 0.5 1.1 — Thyroid 1125.4 2932.7 448.2 814.1 466.71285.4 753.3 751.4 3463.9 605.9 Olfactory Epithelium 12016.8 11374.811191.7 13841.7 9519.2 10724.4 11764.8 9572.8 9321.0 12205.2 AxillaryNodes (2) 0.5 0.4 0.3 0.3 0.4 0.5 0.3 0.4 1.0 3.1 Liver 0.4 0.8 0.4 0.30.3 0.3 0.3 0.4 0.4 0.4 Kidney 1.0 0.4 0.5 0.6 0.4 0.2 0.6 1.0 1.2 0.5Muscle 0.4 0.3 0.3 0.4 0.4 0.2 0.6 0.6 0.7 0.4 Heart 0.4 0.4 0.5 1.6 0.60.3 2.2 0.2 0.2 0.5 Lung 0.6 1.4 0.7 — 1.0 0.5 2.2 1.5 1.1 0.5 LowerCervical Spinal Cord 0.5 5.3 1.0 2.7 0.3 0.1 3.8 0.4 1.8 0.3 ThoracicSpinal Cord 0.1 0.2 0.2 0.4 0.1 0.1 1.2 0.3 0.6 0.1 Lumbar Spinal Cord0.1 0.1 0.1 0.1 0.1 0.7 0.1 0.1 0.1 0.1 Spinal Dura 1.9 3.3 1.3 4.2 1.12.3 — 0.4 1.5 0.8

The method of the invention delivers the therapeutic agent to the nasalcavity of a mammal. It is preferred that the agent be delivered to theolfactory area in the upper one-third of the nasal cavity and,particularly, to the olfactory neuroepithelium in order to promote rapidand efficient delivery of the agent to the CNS along the olfactoryneural pathway rather than the capillaries within the respiratoryepithelium. The preferred transport of the therapeutic agent, e.g., DFO,to the brain by means of the olfactory and trigeminal neural pathwaysrather than the circulatory system so that the harmful side effects andpotentially short half-life of the agent is not an issue. Further,certain agents may simply be unable due to size to cross the blood-brainbarrier from the bloodstream into the CNS. The preferred method allowsdirect delivery of such molecules to the CNS. The data provided in Table1 above strongly supports the increased efficacy of one embodiment ofthe inventive method.

To deliver the therapeutic agent to the CNS, the agent alone or incombination with other substances as a pharmaceutical composition may beadministered to the olfactory area located in the upper one-third of thenasal cavity. The composition may be administered intranasally as apowered or liquid spray, nose drops, a gel or ointment, through a tubeor catheter, by syringe, packtail, pledget or by submucosal infusion.Optimization of the administration of the therapeutic agent is providedby the various embodiments by applying the agent to the upper third ofthe nasal cavity.

The optimal concentration of the active therapeutic agent willnecessarily depend upon the specific neurologic agent used, thecharacteristics of the patient and the nature of the disease orcondition for which the agent is being used. In addition, theconcentration will depend upon whether the agent is being employed in apreventive or treatment capacity. Further, the stage of a particulardisease or disorder, e.g., early vs. late Alzheimer's disease, maydictate the optimal concentration of the agent.

Exemplary work performed according to one embodiment of the inventivemethod was performed, the results of which are illustrated in FIG. 1.The experimental plan included administration of a 10% solution of DFOin distilled water in three doses of 6 mg each (in 60 μl), one doseevery three hours, directly to the CNS of laboratory rats via anintranasal (IN) (upper third of naval cavity) route followed by a 2-hourmiddle cerebral artery occlusion (MCAO) 48 hours post-DFOadministration. The animals were sacrificed 5 days after MCAO and thebrains removed, sectioned on a brain matrix at 2 mm intervals and thenstained with 2,3,5-triphenyltetrazolium chloride (TTC). Infarct volumeswere measured using NIH Image.

Intranasal DFO reduced infarct volumes by 60% when expressed as eithertotal infarct, cortical infarct, or striatal infarct volume. Brainproteins from olfactory bulb and from striatum were subjected to Westernblot analysis for hypoxia-inducible factor-1α (HIF-1α). See FIG. 1 for abar graph illustrating the results (10) for control animals (C) andpretreated animals (P). The results are shown broken into the cortex(12), striatum (14) and overall total (16) infarct volumes forcontrol/pretreated animals. FIG. 1 illustrates the effectiveness ofpretreating an animal with IN DFO administration of three doses of 10%DFO to the upper one-third of the nasal cavity (10), using theexperimental procedure described above in connection with FIG. 1. Here,the control animals' (C) total infarct volume is 175.93 mm³ (18), whilethe pretreated animals' (P) total infarct volume is 70.57 mm³ (20).Thus, a reduction of 60% in infarct volume is realized by way of thispretreatment regimen.

Quantitation of the Western blot revealed that the amount of HIF-1αprotein present in olfactory bulb and striatum was elevatedapproximately 30- and 20-fold, respectively. Additional brain samplesfrom animals treated with intranasal DFO were generated, total RNA wasisolated from olfactory bulb and striatum, and cDNA was generated usingprimers specific for HIF-1α. The cDNA was subjected to RT-PCR, and theresults suggest that mRNA for HIF-1α was abundant; however, there wereno differences in the concentration of HIF-1α mRNA in samples fromanimals treated with intranasal DFO and their controls treated withintranasal distilled water.

The data thus indicate that intranasal DFO pretreatment protects thebrain during stroke via a mechanism involving the transcription factorHIF-1α and that infarct volume is significantly reduced as a result.

With reference to FIG. 2, data indicating the effectiveness ofpretreating an animal with IN DFO administration of one dose of 10% DFOto the upper one-third of the nasal cavity (22), using the experimentalprocedure described above is illustrated. As illustrated, the totalinfarct volume is reduced, as compared with controls treated withdistilled water. The control animals' (C) total infarct volume ismeasured at 117.28 mm³ (24), while the pretreated animals' (P) infarctvolume is 86.72 mm³ (26), an overall infarct volume reduction of 26%.

Referring now to FIG. 3, data indicating the effectiveness ofpretreating an animal with IN DFO administration of three doses of 3%DFO to the upper one-third of the nasal cavity, using the experimentalprocedure described above is illustrated (30). Here, the controlanimals' (C) total infarct volume is 157.01 mm³ (34), while thepretreated animals' (P) total infarct volume is measured to be 101.83mm³ (38), a reduction of 35%.

As the data presented in FIGS. 1-3 indicate, preconditioning animals byadministering IN DFO, or other metal chelator, to the upper one-third ofthe nasal cavity significantly reduces total infarct volume. The Figuresfurther indicate significant reduction of infarct volumes located in theCortex and Striatum.

Thus, in one embodiment of the invention, an effective amount or dose ofa metal chelator may be administered intranasally to precondition theCNS to protect a mammal against the potential of cerebral ischemiaresulting from, e.g., coronary artery bypass graft (CABG) surgery. Theadministration of an effective amount of a metal chelator in the upperone-third of the nasal cavity eliminates the unwanted and potentiallylethal side effects of DFO, for example, possible shock and rapidelimination, while providing the agent with virtually instant access tothe CNS. Other metal chelating agents may have similar unwanted systemiceffects. The metal chelator acts to increase the HIF-1α subunitconcentration and stability of the HIF-1α subunits in the CNS. In thismanner, the metal chelator performs to condition, or precondition, theCNS in anticipation of possible cerebral ischemia resulting from theCABG surgery; establishing a neuroprotective state against a futureepisode of cerebral ischemia.

Several neurologic disorders may be prevented, or the effects minimized,using different embodiments of the inventive method. For example,patients at risk for Alzheimer's disease may be aided by the technique,as this disease involves neurodegeneration, preceded by cerebralvascular difficulties. See, e.g., The Lancet Neurology, vol. 3, pp.184-190, Jack C. de la Torre (March, 2004). Thus, patients at risk forAlzheimer's disease may be pretreated using one or more of the inventiveembodiments disclosed herein.

Further, in another embodiment, those patients scheduled for coronaryartery bypass graft (CABG) surgery may also benefit due to therelatively high percentage of post-surgical cerebral ischemia.

In another embodiment, patients at risk for Parkinson's disease maybenefit from the inventive method.

In yet another embodiment patients at risk for stroke may be aided bythe inventive method. Such patients would include those having riskfactors comprising hypertension, diabetes, obesity, smoking,antiphospholipid syndrome or with a history of stroke (thus prone tosubsequent stroke).

The above embodiments essentially focus on prevention of the cognitive,behavioral and physical impairment due to cerebral ischemia as a resultof certain episodes, disorders or medical procedures by pretreatmentusing IN administration of a metal chelator, e.g., DFO to the upperone-third of the nasal cavities. A series of alternate embodiments focuson treating and/or minimizing the effects of such disorders after theyhave been diagnosed.

In this regard, let us now turn to FIG. 4. Here, the experimental methodis as follows: The method is the same as described above in connectionwith FIG. 1, except that the subjects are not pretreated. Instead, INDFO is administered to the upper one-third of the nasal cavity at thestart of reperfusion to treat the subjects. Six doses of 10% DFO (6 mgDFO/dose) were administered in this manner; three doses of 10% DFO at2-hour intervals on the day of surgery and the remaining three doses of10% DFO at three-hour intervals on the day following surgery (40).Examination of the patient brains reveals that the total infarct volumewas 257 mm³ (42) in the control animals (C) (treated with distilledwater) and 116 mm³ (44) in animals pretreated with IN DFO (P); areduction of 55%.

For example, one embodiment of the inventive method may be used to treata patient having, or recently having undergone, a stroke.

In another embodiment, the inventive method may be used in a treatmentplan for patients at risk for, or diagnosed with, Alzheimer's disease.

In another embodiment, the inventive method may be used to treatpatients at risk for, or diagnosed with, Parkinson's disease.

In another embodiment, the inventive method may be used to treatpatients at risk for, or diagnosed with, Wilson's disease.

In another embodiment, the inventive method may be used to treatpatients at risk for, or diagnosed with, traumatic brain injury, spinalcord injury or cerebral hemorrhage.

In yet another embodiment, patients at risk for, or diagnosed with,stroke and/or transient ischemic attack, and thus at risk for asubsequent stroke, may benefit from the inventive method.

FIGS. 5-8 illustrate another embodiment of the inventive method. Patientweight loss following an ischemic episode is a nagging problem that mayultimately inhibit and slow the patient's recovery time. FIG. 5illustrates the effect of pretreating a patient with administration ofone dose of 10% IN DFO (6 mg DFO/dose) to the upper one-third of thenasal cavity has on patient weight loss post-stroke (50) using theexperimental method described above in connection with FIG. 1. Here, thecontrol subjects (C) (treated with distilled water) lost a total of32.31 grams (52) compared with the subjects pretreated with IN DFO (P)with a weight loss of 4.60 grams (54); an 86% decrease in weight loss.

FIG. 6 illustrates the effect of pretreating a patient withadministration of three doses of 3% IN DFO (6 mg DFO/dose) to the upperone-third of the nasal cavity has on patient weight loss post-stroke(60) using the experimental method described above in connection withFIG. 1. Here, the control subjects (C) (treated with distilled water)lost a total of 54.8 grams post-stroke (62) compared with the subjectspretreated with IN DFO (P) with a post-stroke weight loss of 20.5 grams(64); an 62.6% decrease in weight loss.

FIG. 7 illustrates the effect of pretreating a patient withadministration of three doses of 10% IN DFO (6 mg DFO/dose) to the upperone-third of the nasal cavity has on patient weight loss post-stroke(70) using the experimental method described above in connection withFIG. 1. Here, the control subjects (C) (treated with distilled water)lost a total of 55.38 grams post-stroke (72) compared with the subjectspretreated with IN DFO (P) with a post-stroke weight loss of 8.44 grams(74); an 84.8% decrease in weight loss.

FIG. 8 illustrates the effect of administering IN DFO post-stroke, asopposed to the pretreatment regimes provided in FIGS. 6-8. Here, sixdoses of 10% IN DFO (6 mg DFO/dose) are administered to the upperone-third of the patient's nasal cavity (80). Following stroke, thecontrol subjects (C) (treated with distilled water) lost a total of56.83 grams (82) compared with the subjects pretreated with IN DFO (P)with a weight loss of 44.67 grams (84); an 21.4% decrease in weightloss.

DFO is a particular example of an iron chelator that may be used in oneembodiment to stimulate and/or stabilize HIF-1α to achieve the desiredneuroprotective result. Other iron chelators that may be administeredaccording to an embodiment of the method comprise compounds from thehydroxamate family, and salicylaldehyde isonicotinoyl hydrazone. Otherequivalent iron chelating compounds will present themselves readily tothose skilled in the art and are within the scope of the disclosure.

Alternatively, copper chelating compounds may be administered accordingto an embodiment of the method and comprise trientine, n-acetyl cysteineamide, tetrathiomolybdate and bi-pyridyl compounds. Other equivalentcopper chelating compounds will present themselves readily to thoseskilled in the art and are within the scope of the disclosure.

In another embodiment, a pharmaceutical composition may be comprised ofa combination of at least one iron-chelating compound. Anotherpharmaceutical composition may comprise a combination of at least onecopper-chelating compound. Yet another embodiment of a pharmaceuticalcomposition according to the method may comprise a combination of atleast one iron-chelating compound coupled with at least onecopper-chelating compound.

In still another embodiment, a pharmaceutical composition may becomprised of a combination of at least one metal-chelating compound withat least one antioxidant.

In another embodiment, a pharmaceutical composition comprised of DFO andIGF-1 may be administered.

In general, any of the therapeutic agents or pharmaceutical compositionsdescribed or referenced herein may be administered to patients orsubjects under embodiments of the inventive method prior to a surgicalprocedure such as CABG, during such a procedure or after such aprocedure.

Preferentially, the inventive method, and embodiments thereof, focuseson chelating iron and/or copper. This chelation strategy thus preventscycling of iron and/or copper between an oxidized and a reduced state.Such cycling is highly undesirable as free radicals are formed. Freeradicals and other reactive oxygen species, e.g., H₂O₂, HOCl andradicals such as O₂ ⁻, sulfur cation, nitric oxide radical, ferryl,peroxyl, peroxynitrite, thiyl, thiylperoxyl and alkoxyl, are highlyreactive and may be highly damaging to cellular components as the freeradicals react. Free radical reactions may crosslink proteins, mutateDNA and peroxidize lipids. Such reactions have deleterious effects oncellular homeostasis. Thus, controlling the iron and copper ions throughchelating agents reduces or eliminates such free radical damage from theoxidation/reduction cycling.

As a result, virtually any compound that prevents the cycling of ironand copper between the oxidized and reduced state may be used indifferent embodiments of the inventive method.

In still another embodiment, the therapeutic agent according to theinventive methods may comprise one or more of the following substanceswhich stimulate and/or stabilize HIF-1α: insulin, IGF-I, heregulininsulin, heregulin, TGFbeta, IL-1beta, TNFalpha, cobalt, pyruvate,oxalacetate and lactate. It is within the scope of invention to create apharmaceutical composition combining one or more of the foregoingsubstances. In addition, in other embodiments, the invention mayadminister a pharmaceutical composition comprising at least one of theforegoing substances with at least one metal chelator. Further, apharmaceutical composition may be comprised in another embodiment of atleast one of the foregoing substances combined with at least oneantioxidant.

An effective amount, as herein defined, of the therapeutic agent to beadministered pursuant to embodiments of the invention is the mostpreferred method of expression of dosage. Such effective amount isdependent upon many factors, including but not limited to, the type ofdisease or condition giving rise to an anticipated cerebral ischemicepisode, the patient's general health, size, age, and the nature oftreatment, i.e., short-term of chronic treatment. For illustrativepurposes only, exemplary treatment regimens relating generally to thetherapeutic agents disclosed herein, including dosage ranges, volumesand frequency are provided below:

Efficacious dosage range: 0.0001-1.0 mg/kg.

A more preferred dosage range may be 0.005-1.0 mg/kg.

The most preferred dosage range may be 0.05-1.0 mg/kg.

The dosage volume (applicable to nasal sprays or drops) range may be0.015 ml-1.0 ml.

The preferred dosage volume (applicable to nasal sprays or drops) rangemay be 0.03 ml-0.6 ml.

Generally, the treatment may be given in a single dose or multipleadministrations, i.e., once, twice, three or more times daily over aperiod of time. For chronic disorders such as those diagnosed with, orat risk for, Alzheimer's disease, stroke or Parkinson's disease, thetreatment may consist of at least one dose per day over an extendedperiod of time. Alternatively, for those patients anticipating CABGsurgery, the treatment may be a one-time dose to precondition the CNS inanticipation of potential cerebral ischemia. Such preconditioning mayrequire more than one dose and may be administered from 12 hours to 1week prior to the CABG surgery. Post-stroke treatment may require morethan one dose which may be administered several times over the course ofa day, wherein this treatment regimen may encompass a week or more.

The brain concentrations that are likely to be achieved with the dosageranges provided above are, for a single dose: 0.1 nM-50 μM. Over thecourse of a multi-dose treatment plan, the maximum brain concentrationmay be as high as 500 μM.

The present invention should not be considered limited to the particularexamples described above, but rather should be understood to cover allaspects of the invention. Various modifications, equivalent processes,as well as numerous structures to which the present invention may beapplicable will be readily apparent to those of skill in the art towhich the present invention is directed upon review of the presentspecification.

1. A method to treat patients with neurodegeneration caused by cerebralischemia comprising: administering at least one effective dose ofdeferoxamine (DFO) to the upper one-third of the patient's nasal cavity;and treating the patient with neurodegeneration caused by cerebralischemia.
 2. The method of claim 1, wherein the neurodegeneration isassessed by the size of infarct volume in the patient's brain.
 3. Themethod of claim 1, further comprising administering the at least oneeffective dose of DEG that is within the range of 0.0001 to 1.0 mg/kg.4. The method of claim 3, wherein a more preferred effective dose rangeis 0.005 to 1.0 mg/kg.
 5. The method of claim 1, further comprisingadministering the at least one effective dose of DFO to the upperone-third of the patient's nasal cavity in nasal spray or nasal dropform and wherein the dosage volume is in the range of 0.015 ml to 1.0ml.
 6. The method of claim 5, wherein a more preferred dosage volume isin the range of 0.03 ml to 0.6 ml.
 7. The method of claim 1, furthercomprising administering the at least one dose of DFO until theconcentration of DFO in the patient's brain is within the range of 0.1nM to 50 μM.
 8. A method to pre-treat patients at risk for developingneurodegeneration caused by cerebral ischemia, the method comprising:administering at least one effective dose of deferoxamine (DFO) to theupper one-third of the patient's nasal cavity; and pre-treating thepatient at risk for developing neurodegeneration caused by cerebralischemia.
 9. The method of claim 8, further comprising administering theat least one effective dose of DFO that is within the range of 0.0001 to1.0 mg/kg.
 10. The method of claim 9, wherein a more preferred effectivedose range is 0.005 to 1.0 mg/kg.
 11. The method of claim 8, furthercomprising administering the at least one effective dose of DFO to theupper one-third of the patient's nasal cavity in nasal spray or nasaldrop form and wherein the dosage volume is in the range of 0.015 ml to1.0 ml.
 12. The method of claim 11, wherein a more preferred dosagevolume is in the range of 0.03 ml to 0.6 ml.
 13. The method of claim 8,further comprising administering the at least one dose of DFO until theconcentration of DFO in the patient's brain is within the range 0.1 nMto 50 μM.